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Cardiovascular Research 2000 45(2):370-378; doi:10.1016/S0008-6363(99)00361-2
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Copyright © 2000, European Society of Cardiology

Mechanoelectric feedback after left ventricular infarction in rats

Irina Kiselevaa,e, Andre Kamkina,e, Kay-Dietrich Wagnera,*, Heinz Theresb, Axel Ladhoffc, Holger Scholza, Joachim Günthera and Max J. Labd

aInstitute of Physiology, Humboldt-University (Charité), Tucholskystrasse 2, 10117 Berlin, Germany
bClinic Internal Medicine I, Humboldt-University (Charité), Schumannstrasse 20/21, 10117 Berlin Germany
cInstitute of Pathology, Humboldt-University (Charité), Schumannstrasse 20/21, 10117 Berlin, Germany
dNational Heart and Lung Institute, Imperial College School of Medicine, Charing Cross Hospital, London W6 8RF, UK
eDepartment of Physiology, Martin-Luther-University of Halle, Magdeburger Strasse 6, 0697 Halle/Saale, Germany

* Corresponding author. Tel.: +49-30-2802-6562; fax: 49-30-2802-6662 kay-dietrich.wagner{at}charite.de

Received 2 June 1999; accepted 24 September 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. Methods: Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n=26) or after sham operation (SO, n=16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. Results: Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 µm increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 µm, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 µM) normally used to inhibit stretch-activated channels. Conclusion: After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

KEYWORDS Arrhythmia (mechanisms); Infarction; Ion channels; Membrane potential; Stretch/m-e coupling


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Myocardial infarction (MI) results in structural, biochemical, and electrophysiological alterations in the infarcted as well as in the non-infarcted regions of the heart — a process known as remodeling [1,2]. In the chronic phase following left ventricular MI, scar formation in the infarcted area develops [1]. The left ventricular (LV) volume at low distending pressures is greater than that of non-infarcted controls, independent of the infarct size and the time post-MI [1]. Early structural changes include thinning and lengthening of the infarcted region as well as dilation and hypertrophy of the viable region which start early after MI and continue beyond scar formation.

The properties of the non-ischemic myocardium after MI may be inhomogeneous. Remodeling after infarction is accompanied by severe hypertrophy of the myocytes [3] and by dysfunction of regions adjacent to the scar [4–6]. The more distant zones show specific and complex responses to the mechanical, hormonal, and/or neurohumoral stimuli triggered by the remodeling of the LV [7].

Action potential (AP) characteristics of surviving hypertrophied myocytes are generally altered and more inhomogeneous than in healthy myocardium [8]. In line with this, hypertrophied myocardium has been shown to exhibit electrophysiological properties making it more sensitive to arrhythmias than normal tissue [9,10]. An attractive explanation for the generation of arrhythmias is provided by the mechanoelectric feedback [11–13] which is thought to modulate the electrophysiological properties of cardiac myocytes in response to mechanical stimuli. Mechanoelectric feedback has been shown in a variety of preparations from healthy hearts [14–16], and may involve transmembrane cation fluxes through stretch-activated ion channels (SACs) which in turn may trigger the generation of APs [17–19]. Augmented load on residual myocardium after MI may activate SACs and thereby causing arrhythmia due to electrical instability of cardiac myocytes [11,13,20].

The pathophysiological relevance of altered AP characteristics related to SACs in cardiac tissue after MI needs to be addressed. In view of the observation that changes in load can induce arrhythmia, which is accentuated in diseased hearts [21] possibly due to enhanced mechanoelectric feedback, we propose the following concept: firstly, mechanoelectric feedback is operating not only in intact myocardium, but also in post-MI cardiac tissue; secondly, this feedback is enhanced in post-MI myocardium as compared to normal cardiac muscle; and finally, the mechanoelectric feedback produces arrhythmogenic electrophysiological changes, which involve the operation of stretch-activated ion channels.

The purpose of this study was to examine this hypothesis by analysing the effect of mechanical stretch on electrophysiological characteristics of cardiac myocytes in preparations from infarcted LV of rats. In particular, we examined the role of gadolinium in the mechanoelectric feedback response of the myocardium to induced stretch.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Induction of myocardial infarction
The experiments were performed in accordance with the principles in The Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).

Male Wistar rats (10–15 weeks old), ranging between 220 and 300 g body weight were anaesthetised by intraperitoneal injection of ketamine hydrochloride 75 mg/kg (Ketanest®, Sanofi, Düsseldorf, Germany) mixed with xylazine hydrochloride 7.5 mg/kg (Rompun®, Beyer, Leverkusen, Germany). Limbs were attached to record the electrocardiogram (leads I, II, III, aVR, aVL, aVF). The animals were intubated, and artificial breathing with air was maintained throughout the surgical procedure. Myocardial infarction (group MI) was induced by ligation of the left coronary artery (LAD) as described previously [22,23]. Briefly, we performed a left-sided thoracotomy at a distance of 0.5 cm from the sternum. The heart was exteriorised and the LAD ligated between the outflow tract of the pulmonary artery and the left atrium. Successful ligation of the coronary artery was verified by the occurrence of arrhythmia and ST-segment elevation in the ECG and, visually, by regional myocardial cyanosis. The heart was repositioned and the thorax closed immediately by a previously placed purse-string suture. The skin was closed by a separate suture with the whole procedure taking about 10 min. After recovering from anaesthesia, 20 mg of gentamycinsulfate (Refobacin®, Merck, Darmstadt, Germany) per rat were injected subcutaneously. A 40% mortality within the first 24 h of coronary ligation was observed. The surviving rats were housed, one per cage, on a 12-h light–dark cycle and allowed free access to standard rat chow and water. Sham operated animals (group SO) underwent the identical surgical procedure, except for the coronary artery ligation.

2.2 Experimental preparation and solutions
Five weeks after operation, the rats were anaesthetised with ether, decapitated, and the chest cavity was opened. The hearts were excised and the infarct size was determined by planimetric measurement of the scar tissue on the left ventricular endocardial circumference. Weights of the left ventricle, and the right atrial free wall were determined. For the experiments, we used a standard slice from the left ventricle (8x4 mm), consisting of 50% scar and 50% adjacent unscarred tissue (group MI, n=26). Cardiac myocytes which were located next to the scar tissue were investigated. For comparison with sham operated animals, we selected cells from a comparable region of the left ventricle (group SO, n=16).

Hearts were removed quickly and slices were dissected in oxygenated saline solution. The slices were put into a thermostatically controlled (37°C) constant-flow perfusion chamber with the endocardial surface up. The perfusate for the experiments with stretch and relaxation contained (in mmol/l): NaCl 118, KCl 2.7, CaCl2 1.2, MgSO4 1.2, NaH2PO4 2.2, NaHCO3 25, glucose 5 (pH 7.4). This solution was bubbled with carbogen (5% CO2–95% O2). To avoid precipitation, an oxygenated physiological salt solution [24] was used consisting of (in mmol/l): NaCl 137, KCl 5.4, CaCl2 1.0, MgCl2 0.5, Hepes 5.0, glucose 5.5 (pH 7.4), in the experiments with Gd3+, a blocker of stretch-activated ion channels. This solution was bubbled with O2. No differences in mechanical and electrophysiological properties of the preparations were found between the different solutions. Approximately 90% of the preparations from MI exhibited spontaneous contractile activity. To study stretch-induced electrophysiological phenomena under comparable experimental conditions, only preparations with spontaneous electrical/mechanical activity were used in the MI group. Since we did not observe spontaneous activity in control experiments, preparations in SO were electrically stimulated. Experiments including stepwise stretching and relaxation were performed on 16 preparations from SO and 23 spontaneously contracting tissue specimens from MI. Six of the preparations from SO and nine from MI were subjected to stretch in the presence of 40 µmol/l Gd3+. These preparations were perfused during the entire experiment with the solution described above for the application of Gd3+ allowing us to differentiate between the effects of the higher K+ concentration in this solution and Gd3+ on stretch-induced electrophysiological abnormalities.

2.3 Force measurement and stretch of the tissue
We measured load-dependent force development (F) using the Plugsys system 603 (Hugo Sachs-Elektronik, Germany). The preparations were fixed horizontally between the force transducer and a micrometer, which was used for the adjustment of stretch and preload. The adjusted resting force was not comparable in both groups because one-half of the standard preparation from post-infarcted ventricular myocardium consisted of scar tissue with undefinable visco-elastic properties. Furthermore, the preparations in the MI group did not exhibit a physiological force–length relationship. In an attempt to standardise the mechanical test stimulus, we used the increase in isometric force amplitude due to the applied stretch (Starling mechanism) as an indirect measure. This stimulus was used to provoke the electrophysiological response of the myocyte. This approach was also used for the statistical analysis. For additional indirect quantification and confirmation of the mechanical stimulus, the lengthening combined with stretch was measured with the micrometer.

The preload was adjusted to produce stable force development of 0.5 mN [25]. To simulate changes in enddiastolic pressure/volumes in pathological states, we imposed a sustained diastolic stretch (length changes). Clearly, the degree of stretch to which individual cells of the tissue were subjected will have been different — a situation comparable to studies in situ.

2.4 Electrical stimulation of the preparations in control experiments
Since the SO preparations did not contract spontaneously, we performed electrical stimulation at 0.5 Hz (Plugsys System 603), which was comparable to the range of spontaneous frequency in MI. The biphasic rectangular pulses of 10 ms duration with a current amplitude between 5 and 10 mA did not produce stimulus artefacts distorting the fast upstroke of the AP. Under these conditions, preparations displayed stable contractile activity and electrophysiological parameters during the entire 2-h experimental protocol.

2.5 Electrophysiological measurements
Cellular electrical activity was recorded as described earlier [25]. Briefly, to form a floating microelectrode, a short Ag/AgCl pin connected to the headstage of the amplifier via a platinum–iridium wire with a diameter of 30 µm was partly inserted into a glass microelectrode containing 2.5 mol/l KCl (tip resistance of 50 M{Omega}). This recording electrode and the reference electrode, which was similar, were connected through a high input impedance amplifier (current-clamp scheme).

2.6 Signal processing
The force signal, the amplitude of active force, as well as the electrophysiological signals were fed into an oscilloscope (Nicolet Pro10, Nicolet, USA) and a digital tape recorder (DTR-1801 Biologic, France). Data were digitised and transferred into a personal computer. Resting membrane potential (RMP), action potential (AP) amplitude, AP duration at 25% (APD25), at 50% (APD50), and at 90% of repolarization (APD90) from myocytes with a stable resting membrane potential were evaluated.

2.7 Statistics
Values are given as mean±S.E.M. Significances were determined by the two-tailed Student's t-test for paired and unpaired samples. Significance was assumed at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Five weeks after ligation of the left coronary artery, rats who had undergone the procedure had an infarct size of 16.5±0.6% of the left ventricular endocardial circumference. Ventricular hypertrophy is indicated by increases in left ventricular weight indexed to body weight in MI vs. SO (3.12±0.15 mg/g vs. 2.23±0.06 mg/g, P<0.0001).

Approximately 90% of the MI preparations generated spontaneous APs and contractile activity, which was never observed in the SO group. Examples for APs from SO and MI recorded at preload are depicted in Fig. 1. During the entire experimental protocol, in the absence of any alterations in preload, AP configuration remained stable. Preload was adjusted to a level allowing active force development of 0.5 mN in both groups (Table 1). Post-infarction remodeling resulted in alterations in AP characteristics of the ventricular cardiomyocytes compared to SO (Table 1). Cardiomyocytes in MI had a comparable AP amplitude, a more negative resting membrane potential (–95.2±1.3 mV vs. –88.6±0.8 mV, P<0.005), and in the mean a prolonged AP duration at progressing levels of repolarization (APD90: 129±15 ms vs. 86±3 ms, P<0.05) when compared to SO. In MI vs. SO, greater values of S.E.M. at a greater number of experiments might reflect increased heterogeneity in AP configuration in the ventricular myocardium adjacent to the scar tissue (Fig. 1).


Figure 1
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  		Fig. 1 Continuous recordings of force (F) and action potentials (AP) from the sham operated group (A, n=16) and from the group with MI (B, n=23) and single APs from the SO group (C) and from the group with MI (D, E). Note that the ventricular preparations after sham operation were stimulated electrically whereas preparations after MI generated spontaneous electrical and contractile activity. The different action potential configuration in (D) and (E) points to the increased heterogeneity after infarction.

 

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  		Table 1 Stretch-modulated contractile activity and action potential characteristics of ventricular preparations from sham operated and infarcted rats

 
In both groups, long-lasting stretch led to an increase in active force development providing an intensified mechanical stimulus for the induction of electrophysiological alterations. In SO, stretch by more than 150 µm increased isometric force amplitude from 0.48 to 0.88 mN. In MI, lengthening by only 20 µm increased the isometric peak force from 0.5 to 0.57 mN due to the high stiffness of the preparations. However, effective stretch on the myofilaments will have been different in MI and SO (see above). Extensive stretch in SO and far lower stretch in MI induced a more than twofold increase in APD90 (Table 1). These effects were completely reversible after offset of stretch. Resting membrane potential, as well as AP amplitude were not affected by stretch.

Fig. 2 illustrates the typical effect of long-lasting stretch of ventricular preparations from SO leading to an increase in active force. APD25 and APD50 remained constant during the stretch, whereas APD90 was significantly increased (see also Table 1). The increase in APD90 was associated with stretch-activated depolarization (SAD) which appeared as ‘hump-like’ early after-depolarization.


Figure 2
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  		Fig. 2 Response of cardiomyocyte membrane potential to artificial long-lasting stretch of the left ventricular preparation from the sham operated rats. The increase in stretch (indicated by {uparrow}) produced a stretch-activated depolarization. Offset of stretch (indicated by {downarrow}) led to reversion of this effect. The single registrations were obtained from our continuous recordings 15 s after each step of stretch or release of stretch.

 
Fig. 3 shows typical effects of increases in active force development due to stepwise increases in long-lasting stretch amplitude which prolonged APD90 by growing stretch-activated after-depolarization in SO. Exposure to 40 µM [19,24] Gd3+ only insignificantly reduced the active force by 5% but completely suppressed SADs near to APD90 within 10 min of exposure. Increasing the concentration of Gd3+ to 80 µM had a similar effect which became apparent after 5 min of exposure already (not shown). The resting membrane potential and the AP amplitude were unaffected by Gd3+.


Figure 3
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  		Fig. 3 Example for the suppression of stretch ({uparrow}) activated depolarisations near to APD90 in a left ventricular preparation from the sham operated group by application of 40 µM Gd3+ (n=6). The single registrations were obtained from our continuous recordings 15 s after each step of stretch. Registrations on the effect of Gd3+ were made after 5 and 10 min of application.

 
In contrast to SO, where a high degree of stretch increased active force development by 80%, a minor stretch which increased the active force amplitude by only ~15% clearly evoked SADs in MI. However, from the clearly different increase in active force due to stretch we cannot exactly estimate the degree of stretch affecting the membranes of individual cells since we do not know what the stretch is doing to the sarcomere spacing.

In the cases of relatively short APD, the SADs appeared near to APD90 (n=16, for example see Fig. 4). In the cases of extremely prolonged APs, SAD was already observed close to APD50 (n=7, for example see Fig. 5) at comparable degrees of stretch as in the experiments with SADs near to APD90. The relationship between stretch-induced ‘hump-like’ SAD near to APD50 and the degree of stretch is shown for the MI group. Small degree of stretch led to a small amplitude of the SAD. Further stretch led to an increase in the SAD. The increases in APD90 or APD50 after stretch were due to the development of SADs. Offset of stretch demonstrated the complete reversibility of alterations in AP configuration due to stretch-activated depolarizations.


Figure 4
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  		Fig. 4 Example for the response of cardiomyocyte membrane potential to artificial long-lasting stretch of a left ventricular preparation in the group with myocardial infarction. The increase in stretch (indicated by {uparrow}) led to stretch-activated depolarization near to APD90. Offset of stretch ({downarrow}) resulted in complete reversibility of the stretch-induced effect (n=16). The single registrations were obtained from our continuous recordings 15 s after each step of stretch or release of stretch.

 

Figure 5
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  		Fig. 5 Response of cardiomyocyte membrane potential to artificial long-lasting stretch of a left ventricular preparation in the group with myocardial infarction. The increase in stretch (indicated by {uparrow}) led to stretch-activated depolarization near to APD50. Offset of stretch ({downarrow}) resulted in complete reversibility of the stretch-induced effect (n=7). The single registrations were obtained from our continuous recordings 15 s after each step of stretch or relaxation.

 
Fig. 6 depicts the effects of Gd3+ on active force development and AP configuration in MI. The stretch-induced afterdepolarizations at APD90 were suppressed completely after 10 min of application of 40 µM Gd3+ as were those near to APD50 (not shown for MI). Increasing the concentration of Gd3+ to 80 µM led to a similar effect already after 5 min. The resting membrane potential, the AP amplitude, and the frequency of spontaneous contractions were unaffected by Gd3+ exposure as already seen in the experiments in the SO group where SADs appeared near to APD90.


Figure 6
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  		Fig. 6 Example for the suppression of stretch ({uparrow}) activated depolarisations near to APD90 in a left ventricular preparation from the group with myocardial infarction by application of 40 µM Gd3+ (n=9). The single registrations were obtained from our continuous recordings 15 s after each step of stretch. Registrations on the effect of Gd3+ were made after 5 and 10 min of application.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We show that (i) ventricular tissue samples from the infarction borderzone generate spontaneous contractile activity; (ii) APs from rat ventricular myocytes adjacent to the scar after MI are prolonged and have a greater variability compared to SO; (iii) increased stretch induces proportional and reversible changes in the AP associated with stretch-activated depolarizations; (iv) after MI, the sensitivity to stretch-induced changes in the AP configuration is more pronounced; (v) SADs are suppressed by Gd3+ indicating involvement of stretch-activated channels. The appearance of SADs is consistent with contraction–excitation feedback, or mechanoelectric feedback [16] which has been described by many investigators in different species of healthy animals [13]. This study reports for the first time SADs in multicellular preparations from chronically infarcted rat ventricles.

4.1 Remodeling after myocardial infarction
Remodeling after MI is associated with phenotype alterations and the development of hypertrophy of the surviving myocardium [8]. Accordingly, an increase in myocyte cross-sectional area and length of left ventricular myocytes has been reported [26]. In our experiments, hypertrophy is indicated by the increased LV weight indexed to body weight. Also hypertrophy due to hypertension has been shown to lengthen APD and to increase the dispersion in APD [27].

4.2 Characteristics of action potentials
In unaffected hearts, AP configurations of different cardiomyocytes were relatively invariable in the endocardial region of the ventricle, in which infarcts were set in MI. After MI, AP configurations were more heterogeneous in the myocytes from remodeled myocardium adjacent to the scar tissue and also markedly differed with regard to the kinetics of repolarization as compared to SO. It has been shown [8] that the duration of APs is increased and the time course of repolarization shows marked heterogeneity in myocytes from remodeled LV from rats after MI. The prolongation of APD has been explained by decreased K+ outward currents, rather than by changes in Ca2+ inward currents [8]. The electrophysiological heterogeneity may result from differences in remodeling between individual cells. In line with this, the scar itself as well as the adjacent myocardium are relatively non-distendable and much more resistant to stretch. It is known by the classical description of Tennant and Wiggers [28] that in post-MI hearts mechanical forces deform the hypoperfused non-contracting scar region. These forces act differently on both the necrotic and non-infarcted myocardium, which is of particular importance for the observed electrophysiological heterogeneity in the myocardium adjacent to the scar. The hyperpolarization in resting membrane potential in MI compared to SO is expected to be associated with an increase in AP amplitude which we did not observe. These results probably suggests a reduced Na+ inward current into cardiomyocytes from the borderzone of the infarcted left ventricle as reported for infarcted canine hearts [29].

4.3 Stretch-induced changes in action potential configuration
In SO, extensive stretch led to an increase in APD90 due to the appearance of SADs. In contrast to ‘classical’ early or delayed afterdepolarizations [30,31], the observed SADs did not depend on triggering by the preceding AP. After MI, the lower degree of stretch indicated by the significantly smaller increase in active force compared to SO, induced SADs in the heterogeneous APs near to APD50 or APD90. In this study, SADs did not produce arrhythmia. Stretch-induced generation of arrhythmia has recently been demonstrated in a variety of experiments performed with preparations of healthy whole heart and ventricular preparations [13]. The generation of stretch-induced arrhythmia has been shown to depend on the rate and the intensity of the mechanical stimulus. In our experiments, we used a slow increase in stretch amplitude and a relatively low degree of stretch to provide a more physiological mechanical stimulus. Furthermore, the slow rate of stretch was chosen to avoid mechanical artefacts in the electrophysiological registration due to microelectrode movements. Application of a more intense stretch would possibly have induced arrhythmia also in our preparations.

It has been reported that stretch can shorten, lengthen or not affect the AP configuration, depending on the duration and the timing of the mechanical stimulus [15,32–37]. Stretch-induced changes in the AP configuration may mimic early or delayed afterdepolarizations depending on whether experimental stretch is applied in late systole or early diastole. It has been shown [37] that the amplitude of stretch is more important for the response of the membrane potential than its duration. It has been shown that increased preload caused APD shortening and increase in afterload evoked a delay in final repolarization due to the appearance of early afterdepolarizations [13,37,38]. In our experiments, the applied long stretch of {approx}30 s implicates the close interaction of preload, afterload and contractility. Therefore, characteristics of the applied stretch, e.g. increment or decrement rates, cannot be related to the responses in membrane potential. The SADs, however, were related to the applied long stretch but not to the stress of preparations during active force development, since the far lower increase in stretch-dependent active force in the MI group induced comparable AP changes.

One key result of this study is the observation that in remodeled myocardium adjacent to the scar, stretch elicited SADs more readily compared to ventricular myocardium from SO. In the MI group, the higher apparent sensitivity to stretch is probably not due to the stiffness of the scar area because lower length changes were associated with a proportionally lower increase in active force when compared to SO. The enhanced sensitivity of the membrane potential to stretch may result from interaction with surrounding cells e.g. fibroblasts [19] in the tissue and/or from altered membrane properties of single cardiomyocytes.

4.4 Mechanically-induced electrical artefact?
In early experiments, observed afterdepolarizations were dismissed as a movement artifact by Hoffman et al. [39]. Growing evidence reveals [15,40,41] that stretch-induced afterdepolarizations are true responses of membrane potential to stretch because: (i) resting membrane potentials and AP amplitudes were stable; (ii) changes in AP duration were fully reversible after termination of stretch; (iii) during stretch application, we did not observe typical wave-like fluctuations of the membrane potential which are normally seen when the microelectrode moves out of the cell; (iv) stretch-induced changes in AP configuration could be inhibited by Gd3+, a blocker of stretch-activated channels.

4.5 Involvement of stretch-activated channels
Recently the existence of non-selective SACs has been documented in cardiac cells [19]. Hu and Sachs [42] reported a reversal potential of mechanosensitive currents in cardiac cells in the range of –15 to –18 mV. Bustamante et al. [43] estimated the reversal potential for those SACs, produced mainly by Na+-, K+-, and Ca2+- currents, to be –40 mV. Myocardial SACs can be passed by inward and outward currents [17,43,44]. Under physiological conditions, stretch during the AP plateau where the membrane potential is more positive than the reversal potential, would repolarize the membrane, i.e. shorten the APD by cation efflux. When the membrane potential is more negative than the reversal potential, stretch would induce an inward current to prolong APD and subsequently producing SADs. We suggest that long-lasting stretch in our experiments activated a Gd3+-sensitive inward current through SACs which caused the observed SADs. A recently reported mathematical model including a putative SAC partially predicted the electrophysiological abnormalities we observed in our experiments [45]. These electrophysiological abnormalities were sensitive to Gd3+ which supports strongly the involvement of SACs. It must be noted, that Gd3+, in addition to blocking SACs, has also been reported to inhibit voltage-gated Ca2+ channels in isolated guinea-pig ventricular myocytes [46]. Hansen et al. [38] showed that Gd3+ but not organic inhibitors of Ca2+ channels blocked stretch-induced electrophysiological abnormalities in isolated canine myocardium. Since in our experiments 40 µM Gd3+ did not affect the AP plateau and contractile function of the preparations simultaneously with blocking of SADs, inhibition of voltage-gated Ca2+ channels by Gd3+ appears unlikely to account for our findings. Moreover, since the delay for the observed effects was much less with 80 than with 40 µM Gd3+, it is possible that limited diffusion in the preparations decreased the effective concentration of Gd [3] to a level which was too low to block voltage-operated Ca2+ channels. The delayed negative inotropic effect of Gd3+ might have appeared when the agent had reached higher less specific concentrations after diffusion. We therefore suggest that suppression of SADs by Gd3+ in our experiments was due to inhibition of SACs rather than blockade of voltage-operated Ca2+ channels.

In summary, after MI, SADs are generated in the borderzone of the infarcted area at lower degrees of stretch, which may contribute to the higher risk of ventricular arrhythmia after infarction [21].

4.6 Limitations of the study
From the present study it remains unclear whether the high mechanosensitivity of the membrane potential is typical for surviving myocardium after infarction, or whether it is a common phenomenon in a hypertrophic setting. Clinical observations, however, support the hypothesis of this being a specific feature of surviving dilated myocardium after MI. To clarify this point, further studies on different models of cardiac hypertrophy should be performed. The experiments with the application of Gd3+, which blocked the stretch-induced electrophysiological abnormalities, strongly suggest the involvement of SACs in these phenomena, but we cannot exclude the possibility that other ion channels are also involved in the generation of the observed electrophysiological abnormalities.

Time for primary review 22 days.


   Acknowledgements
 
The experimental studies were supported by the Humboldt University of Berlin and Alexander von Humboldt-Stiftung (Germany). AK was a fellow of the Alexander von Humboldt-Stiftung, IK received a travel grant from the Humboldt University of Berlin.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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